During my biofilm assay, I had forgotten to wash off the excess crystal violet and instead shook out as much as I could. Although this was a mistake, I can still see that the values of the replicates from each extract to be constant to each other. However, I dont think that this is what had caused my results to go sideways as when I compare the results to my positive control, I can see that the optical density is higher in the readings where there was a plant extract as an antimicrobial. Should this be lower than the positive control? I have some suggestions as to why this may be:
-some of the extracts that I used such as guava were pigmented and this could have given a higher optical density reading.
-The plant extracts may have allowed the biofilm formation to thrive better than in the positive control which included only water and broth + bacteria.
Is there any way that I can salvage my results?? Im mainly confused as to why the positive control was lower than with the plant extracts.
Also is the biofilm assay strictly set for an absorbance at 595nm? as for one of my plates, I read at 405nm, giving me higher values compared to 595nm.
Dear Mikhaela,
While I advise you to tread carefully with over-analysing an experiment which you realise has been misperformed, there are some rather exciting answers to why plant extracts might stimulate higher levels of biofilm–formation compared to your control.
First, the extracts may coat the surface of your culture container, providing a better chemical environment which allows greater bacterial colonisation and subsequent biofilm-formation (e.g. we have shown that simply washing a plastic sampling device in PBS or soil water results in a better surface for Pseudomonas attachment).
Second, the extracts may coat the bacteria (or more generally associate with bacterial surface structures) to enable better surface colonisation.
Finally, the extracts may act as a signal for one or more of the processes involved in biofilm–formation (e.g. inhibition of swimming motility, induction of attachment, twitching, EPS production, etc.).
You could look at these possibilities without having to redo your main experiment (which I think really is required): if you wash culture containers with the plant extract, can you detect a coating after it is washed out again (perhaps by using various dyes)? You could consider doing MATH assays with or without pre-extract treatments to see if bacterial cell hydrophobicity changes, and if you have enough plant extract, you could test to see whether swimming and twitching (if your strain does this) are affected.
Finally, I would suggest that your preconceived notions of the effect of the plant extracts (e.g. be useful in controlling biofilm-formation) might be biasing your understanding of what they do - your bacteria may be responding to natural environmental signals (such as plant compounds) which initiate biofilm–formation.
Regards, Andrew
Hello Mikaela,
Biofilm formation rate of microbes depends on the environments and different experimental methods such as microtiter well plate assay. In my viewpoint, you may change the protocol of biofilm formation assay. The crystal violet staining (0.1%) may allow for 30 minutes then wash twice with PBS and dry it at 55 degree Celsius for 30 minutes. I hope it will be helpful to get your desired result.
Dear Mikhaela,
While I advise you to tread carefully with over-analysing an experiment which you realise has been misperformed, there are some rather exciting answers to why plant extracts might stimulate higher levels of biofilm–formation compared to your control.
First, the extracts may coat the surface of your culture container, providing a better chemical environment which allows greater bacterial colonisation and subsequent biofilm-formation (e.g. we have shown that simply washing a plastic sampling device in PBS or soil water results in a better surface for Pseudomonas attachment).
Second, the extracts may coat the bacteria (or more generally associate with bacterial surface structures) to enable better surface colonisation.
Finally, the extracts may act as a signal for one or more of the processes involved in biofilm–formation (e.g. inhibition of swimming motility, induction of attachment, twitching, EPS production, etc.).
You could look at these possibilities without having to redo your main experiment (which I think really is required): if you wash culture containers with the plant extract, can you detect a coating after it is washed out again (perhaps by using various dyes)? You could consider doing MATH assays with or without pre-extract treatments to see if bacterial cell hydrophobicity changes, and if you have enough plant extract, you could test to see whether swimming and twitching (if your strain does this) are affected.
Finally, I would suggest that your preconceived notions of the effect of the plant extracts (e.g. be useful in controlling biofilm-formation) might be biasing your understanding of what they do - your bacteria may be responding to natural environmental signals (such as plant compounds) which initiate biofilm–formation.
Regards, Andrew
Nice discussion about different biofilm formation assays.
To my opinion, without proper washing you can get misinterpreted data which will make you more confused. For clear understanding, you need to repeat it at least twice. You can also check other biofilm assays as suggested.
Hi everyone, I appreciate all of your suggestions. I think it would also be worth mentioning that for P. aeruginosa, I followed the protocol correctly and washed off the excess crystal violet. This showed that the Kelp extract still produced a higher optical density in comparison to the positive control. I do think that it could be to do with the pigments in my extract or the effect of quorum sensing on the bacteria.
Hello,
Do you have blanks with the Kelp extracts and no bacteria?
I have had results showing that at low bactericide concentrations bacteria aggregate and produce more biofilm than just in medium without any bactericide. The formation of biofilm increases the survival of the bacterial population, therefore biofilm formation is an advantage in the presence of antimicrobials.
Here is the link to the paper
Article Influence of selected bactericides on biofilm formation and ...
However if you don't trust your cristal violet staining protocol you may want to observe and compare biofilms on the microscope; fluorescence, SEM, etc.
Good luck!
Marta,
so are you suggesting that if they are at a low bacteriacide concentrations it only aggregates the bacteria to produce more biofilm therefore if I had to do the experiment again, a higher concentration of kelp extract would be more effective?
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